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| United States Patent |
5,780,257
|
|
Aoyama
, et al.
|
July 14, 1998
|
Method and reagent for detecting peroxidase or hydrogen peroxide
Abstract
Method for detecting peroxidase or hydrogen peroxide with high sensitivity.
Both peroxidase and hydrogen peroxide are prepared such that one of them
is overabundant to the other. Phenoxy radicals are produced from a
p-substituted phenol compound by the action of peroxidase in the presence
of hydrogen peroxide. The free radicals are trapped by a hydroxy amine
compound, and stable radical species are produced. Electron spin
resonances of the stable radical species are measured.
| Inventors:
|
Aoyama; Masaaki (Yamagata, JP);
Shiga; Masanobu (Rockville, MD)
|
| Assignee:
|
Yamagata Technopolis Foundation (JP);
JEOL Ltd. (JP)
|
| Appl. No.:
|
683148 |
| Filed:
|
July 18, 1996 |
| Current U.S. Class: |
435/28; 423/582; 423/584; 435/25; 435/968; 436/173; 564/300 |
| Intern'l Class: |
C12Q 001/28; C12Q 001/26; G01N 033/53; G01N 024/00 |
| Field of Search: |
435/6,7,28,25,968
423/582,584
564/300
436/173
|
References Cited
U.S. Patent Documents
| 4828983 | May., 1989 | McClune | 435/7.
|
| 5372931 | Dec., 1994 | Friedman et al. | 435/6.
|
Other References
Fischer et al., "Direct Electron Spin Resonance Detection of Free Radical
Intermediates During The Peroxidase Catalyzed Oxidation of Phenacetin
Metabolites," Chemico-Biological Interactions 1986, 60, 115-127, Nov.
1986.
Ross et al., "The Generation and Subsequent Fate of Glutathonyl Radicals in
Biological Systems," J. Biol. Chem. 1985, 260, 15028-15032, Dec. 5, 1985.
Thorpe, Gary H. G., et al., "Enhancement of the Horseradish
Peroxidate-Catalyzed Chemiluminescent Oxidation of Cyclic Diacyl
Hydrazides by 6-Hydroxybenzothiazoles", Analytical Biochemistry 145,
96-100 (1985), pp. 96-100.
Klebanoff, S. J., "An Effect of Thyroxine and Related Compounds on the
Oxidation of Certain Hydrogen Donors by the Peroxidase System", Journal of
Biological Chemistry, vol. 234, No. 9, Sep. 1959, pp. 2437-2442.
Toshiyuki Ohnishi et al., "One-Electron-Transfer Reactions In Biochemical
Systems", Biochim. Biophys. Acta, 172 (1969) pp. 357-369.
|
Primary Examiner: Leary; Louise
Attorney, Agent or Firm: Webb Ziesenheim Bruening Logsdon Orkin & Hanson, P.C.
Claims
What is claimed is:
1. A method for detecting peroxidase or hydrogen peroxide, said method
comprising the steps of:
preparing peroxidase and hydrogen peroxide such that one of them is
overabundant to the other;
producing phenoxy radicals from a p-substituted phenol compound by the
action of the peroxidase in the presence of the hydrogen peroxide;
transferring electrons from said phenoxy radicals to a hydroxy amine
compound and producing stable radical species of said hydroxy amine
compound; and
measuring electron spin resonances of said stable radical species.
2. The method of claim 1, wherein said p-substituted phenol compound is a
4-acetamide phenol derivative given by
##STR4##
where R.sup.1 is a hydrogen atom or C.sub.1-6 alkyl group, R.sup.2 is
C.sub.1-6 alkyl group, carboxyl, or C.sub.1-6 alkoxy carbonyl group.
3. The method of claim 1, wherein said p-substituted phenol compound is
selected from the group consisting of 4-methoxyphenol, 4-ethoxyphenol,
4-iodophenol, 3-(4-hydroxyphenyl) propionic acid, 4-hydroxyphenylacetic
acid, 4-hydroxyhippuric acid, p-cresol and tyramine.
4. A reagent for detection of peroxidase or hydrogen peroxide, said reagent
consisting of a hydroxy amine compound which can accent electrons
transferred from phenoxy radicals to produce stable radical species of
said hydroxy amine compound, said phenoxy radicals formed by the action of
peroxidase in the presence of hydrogen peroxide and a p-substituted phenol
compound.
5. The reagent of claim 4, wherein said p-substituted phenol compound is a
4-acetamide phenol derivative of the formula
##STR5##
where R.sup.1 is a hydrogen atom or C.sub.1-6 alkyl group, R.sup.2 is
C.sub.1-6 alkyl group, carboxyl, or C.sub.1-6 alkoxy carbonyl group.
6. The reagent of claim 4, wherein said hydroxy amine compound is produced
by reducing a nitroxide compound which can be used as a spin-labeling
agent for free radicals.
7. A hydroxy amine compound capable of accepting electrons transferred from
phenoxy radicals created by the action of peroxidase in the presence of
hydrogen peroxide and a p-substituted phenol derivative and which can
produce stable radical species of said hydroxy amine compound.
8. The method of claim 1 wherein said hydroxy amine compound is produced by
reducing a nitroxide compound which can be used as a spin-labeling agent
for free radicals.
Description
FIELD OF THE INVENTION
The present invention relates to a method of detecting peroxidase or
hydrogen peroxide. The invention also relates to reagents used for the
detection of peroxidase (POD) or hydrogen peroxide (H.sub.2 O.sub.2).
BACKGROUND OF THE INVENTION
Chemiluminescence using chemical reagents, such as luminol, is well known
as a method for detecting peroxidase. A method using p-substituted phenol
derivatives has been proposed in order to improve the sensitivity of the
measurement utilizing chemiluminescence. For example, it has been reported
that chemiluminescence due to luminol-H.sub.2 O.sub.2 -POD is intensified
by p-iodophenol (Kricka, et al., Anal. Biochem., 145, 96, 1985). Luminol
is used as a monoanion complex of luminol in an aqueous solution and is
changed to thiazasemiquinone radical by an oxidative reaction of a
p-substituted phenol compound. The thiazasemiquinone radical is finally
changed to 3-amino phthalic acid via some intermediates. The
chemiluminescence occurs at the final step in the reaction. The iodophenol
(iodophenol radicals) reacts with the luminol and promotes the generation
of thiazasemiquinone radicals. An immunity-measuring reagent named
"Amerlite" used for this principle is available from Nippon Kodak
Diagnosis Techcs Co., Ltd., Japan.
It has been reported that addition of p-methoxyphenol to ascorbic
acid-H.sub.2 O.sub.2 -POD enhances generation of ascorbic radicals
(Chance, B., Arch. Biochem. Biophys., 41, 389, 1952). Similar enhancing
reactions can be produced by using p-cresol (Ohnishi, T. et al., Biochem.
Biophys. Acta, 172, 357, 1969) or thyroxine or its analogues (Klebanoff,
S. J., J. Biol. Chem., 234, pp. 2437-2442, 1972).
A p-substituted phenol derivative induces enhancement of luminol
luminescence and of generation of ascorbic radicals, therefore, it is
useful as an enhancer in detecting peroxidase. In the reaction, phenoxy
radicals produced by peroxidase act on luminol and on ascorbic acid,
causing a one-electron pullout (oxidation) reaction. However, even if a
method making use of such intensified luminescence or radical
amplification is adopted, peroxidase is not detected with sufficient
sensitivity. Especially where peroxidase is detected utilizing radical
amplification, the minimum concentration of detection is about 10.sup.-3
units/ml. It is obvious that the sensitivity is insufficient for a reagent
for enzymatic immunoassay.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a novel method for
detecting peroxidase or hydrogen peroxide with high sensitivity.
It is another object of the invention to provide reagents permitting
high-sensitivity detection of peroxidase or hydrogen peroxide.
Where a p-substituted phenol derivative is used as an enhancer, the
achieved enhanced efficiency of the sensitivity might depend on the life
of phenoxy radicals produced by the action of peroxidase. Accordingly,
applicants have understood that the above-described method is unable to
accomplish a great sensitivity improvement because neither luminol
radicals (or reaction intermediates) nor ascorbic radicals (or final
products) are stable radicals. The applicants have earnestly searched for
an enhancer for producing stable phenoxy radicals and for means for
converting phenoxy radicals into other stable radical species producing
strong signals.
As a result, it has been discovered that where phenoxy radicals produced
from a p-substituted phenol derivative that is an enhancer are oxidized by
a hydroxy amine derivative, stable radical species producing strong ESR
signals are generated. Also, it has been found that peroxidase can be
detected with quite high sensitivity if a p-acetamide phenol derivative is
utilized as an enhancer in employing the above-described hydroxy amine
derivative. Furthermore, it has been found that hydrogen peroxide can be
detected by causing an excessive amount of peroxidase to be present in the
same reaction system. The present invention has been made, based on the
above- described findings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1(a) is an ESR spectrum of a hydroxy amine compound, consisting of
carboxy-PTIOH, oxidized by phenoxy radicals, and in which the upper line
indicates the ESR signal under a blank condition;
FIG. 1(b) is an ESR spectrum of a hydroxy amine compound, consisting of
HHTIO, oxidized by phenoxy radicals, and in which the upper line indicates
the ESR signal under a blank condition;
FIG. 2(a) is an ESR spectrum similar to FIG. 1(a), but showing the
signal-to-noise ratio (S/N) of a measurement performed by a method
according to the invention where the concentration of the peroxidase
solution is 10.sup.-5 u/ml;
FIG. 2(b) is an ESR spectrum similar to FIG. 1(b), but showing the S/N of a
measurement performed by a method according to the invention where the
concentration of the peroxidase solution is 10.sup.-5 u/ml;
FIG. 3 is a chart showing ESR signal waveforms obtained by changing the
concentration of hydrogen peroxide in Example 5;
FIG. 4 is a graph showing the relation of the ESR signal intensity to the
concentration of hydrogen peroxide obtained in Example 5;
FIG. 5(a) is a chart showing ESR signal waveforms derived from a standard
substance in Example 6; and
FIG. 5(b) is a chart showing ESR signal waveforms derived from monitored
control sera in Example 6.
DETAILED DESCRIPTION OF THE INVENTION
The present invention includes a method for detecting peroxidase or
hydrogen peroxide in accordance with the present invention comprises the
steps of: preparing peroxidase and hydrogen peroxide such that one of them
is excessive for the other; producing phenoxy radicals from a
p-substituted phenol compound by the action of the peroxidase in the
presence of the hydrogen peroxide; oxidizing the phenoxy radicals by a
hydroxy amine compound to produce stable radicals; and measuring electron
spin resonances of the stable radical species.
One preferred embodiment of the invention is based on the above method and
characterized in that the p-substituted phenol compound is 4-acetamide
phenol derivative where the p-substituted phenol compound is given by
##STR1##
where R.sup.1 is a hydrogen atom or C.sub.1-6 alkyl group, R.sup.2 is
C.sub.1-6 alkyl group, carboxyl, or C.sub.1-6 alkoxy carbonyl group.
Alternatively, the p-substituted phenol compounds are selected from the
group consisting of 4-methoxyphenol, 4-ethoxyphenol, 4-iodophenol,
3-(4-hydroxyphenyl) propionic acid (HPPA), 4-hydroxyphenylacetic acid,
4-hydroxyhippuric acid, p-cresol and tyramine.
In another embodiment of the invention, a reagent for detection of
peroxidase or hydrogen peroxide consists of a hydroxy amine compound
capable of trapping electrons of phenoxy radicals produced by the
enzymatic reaction of peroxidase under the presence of peroxidase and
hydrogen peroxide and, preferably, the aforementioned 4-acetamide phenol
derivative and of producing stable radical species. Also, a hydroxyamine
compound is offered that is a phenoxy radical-trapping reagent capable of
trapping electrons of phenoxy radicals produced by the action of
peroxidase under the presence of peroxidase and hydrogen peroxide and,
preferably, the aforementioned 4-acetamide phenol derivative and of
producing stable radical species.
The hydroxy amine compound used in the method according to the present
invention is a compound reacting as a reagent for detection of peroxidase
or hydrogen peroxide. This compound traps phenoxy radicals produced by the
action of peroxidase under the presence of hydrogen peroxide and a
p-substituted phenol compound. As a result, stable radical species are
generated. It is to be noted that the present invention does not stick to
any certain theory. These hydroxy amine compounds trap phenoxy radicals
and give rise to stable nitroxide (NO) radical species. These stable
radical species can be quantitatively detected by an electron spin
resonance (ESR) spectrometer. In this way, peroxidase or hydrogen peroxide
can be detected with quite high sensitivity. Detections and measurements
referred to herein embrace qualitative analysis, quantitative analysis and
every kind of detection.
Any kind of hydroxy amine compound can be used as the above-described
hydroxy amine compound used as a reagent for detection of peroxidase or
hydrogen peroxide, as long as the compound can trap electrons of phenoxy
radicals and produce stable radical specimens. It would be easy for those
skilled in the art to make a decision according to the method described in
the body of the present specification as to whether the hydroxy amine
compound has characteristics preferable for the method according to the
invention. Generally, stability of radical species can be judged from
their lives, aging characteristics of ESR signal intensities or other
factors.
Preferred reagents used for detection of peroxidase or hydrogen peroxide
can be hydroxy amine compounds which have the above-described
characteristics and are obtained by reducing nitroxide (nitron) compounds
that can be used as free radical spin-labeling agents. Nitroxide compounds
which can be used as raw materials include: TEMPO
(2,2,6,6-tetramethyl-1-piperidinyloxy), its derivatives, PROXYL
(2,2,5,5-tetramethylpyrrolidinyloxy), its derivatives, carboxy-PTIO
›2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide), its
derivatives, DOXYL (4,4-dimethyl-3-oxazolinyloxy), and its derivatives.
All of these compounds are commercially available (see, for example, a
catalog of Sigma Corporation, 1995 edition, 1943, "SPIN LABELS AND SPIN
TRAPS") and are easy to obtain. Furthermore, nitroxide-based spin labeling
agents as described, for example, by Tokuko Watanabe in the summary of
Dhojin News, No. 1, "Application of Spin Labeling to Biopolymers", 1976,
can be used as nitroxide compounds.
Hydroxy amine compounds which can be preferably used in the novel method
are given by
##STR2##
where R.sup.3 is a hydroxyl group, a substituted or non-substituted amino
group (preferably non-substituted amino group), carboxyl, or C.sub.1-6
alkoxy carbonyl group; R.sup.4 is a hydrogen atom, C.sub.1-6 alkyl group
(preferably methyl group), or hydrazid group; R.sup.5 is hydroxyl group, a
substituted or non-substituted amino group (preferably C.sub.1-6 alkyl
carbonyl-substituted amino group or non-substituted amino group),
carboxyl, or substituted or non-substituted carbamoyl (preferably
non-substituted carbamoyl); R.sup.6 is a hydrogen atom or C.sub.1-6 alkyl
group (preferably methyl group); and R.sup.7 is a hydrogen atom or
C.sub.1-6 alkyl group (preferably methyl group).
Any of the above-described preferred hydroxy amine compounds can be
produced by the reductive reaction from nitroxide compounds which are used
as free radical spin-labeling agents. These hydroxy amine compounds can
trap phenoxy radicals and produce stable radical species. These compounds
can be easily prepared by methods described in papers with or without
appropriate modifications to the starting materials or reagents used in
the methods described in the papers. Furthermore,
4-hydrazomethyl-1-hydroxy-2,2,5,5-tetramethyl-3-imidazoline-3-oxide
(HHTIO) is commercially available (Aldrich Corporation, Code No. 33,
114-7). If necessary, the compounds can be refined or diluted in using
them.
A mild reducing agent such as ascorbic acid can be used to reduce a
nitroxide compound which is a raw material compound. According to the used
method of reduction, the hydroxy amino group may be further reduced and
changed into a compound (RR'NH) having an amino group. Since this amino
compound is not detected by ESR spectroscopy, if a small amount of such
amino compound is introduced in the novel reagent for detection of
peroxidase or hydrogen peroxide, no practical problems take place.
Generally, the purity of a hydroxy amine compound can be calibrated by
.sup.1 H-NMR measurements.
In the novel method, two or more of the above-described hydroxy amine
compounds may be used in conjunction. Hydroxy amine compounds which can be
especially preferably used as reagents for trapping electrons of phenoxy
radicals and as reagents used for detection of peroxidase or hydrogen
peroxide are given below. It is to be noted, however, that reagents used
for detection are not limited to these examples:
##STR3##
P-substituted phenol compounds which are used in the novel method should be
phenol derivative compounds having arbitrary substituted groups at least
in the p-position. As decomposition of hydrogen peroxide in the presence
of a catalyst consisting of peroxidase progresses, the compound should be
readily converted into corresponding phenoxy radicals. As long as these
requirements are met, any desired kind of p-substituted phenol compound
can be used. In the method according to the present invention, such a
p-substituted phenol compound acts as an enhancer in a decomposition
reaction of hydrogen peroxide in the presence of peroxidase, or a
catalyst. Those skilled in the art can make a decision according to the
method described in the body of the present specification as to whether
the p-substituted phenol compound has characteristics described above.
The above-described p-substituted phenol compounds may have substituted
groups in the m- and/or p-positions. No limitations are imposed on the
kind of the substituted group in the p-position existing in the benzene
rings of p-substituted phenol compounds or on the kinds of one or more
substituted groups not in the p-position. Where two or more substituted
groups exist, they may be similar or dissimilar. Examples of these
substituted groups include C.sub.1-6 alkyl group, C.sub.1-6 alkenyl group,
C.sub.1-6 alkinyl group, C.sub.1-6 alkoxy group, halogen atoms,
halogenated C.sub.1-6 alkyl group, carboxyl group, carboxy C.sub.1-6 alkyl
group, C.sub.1-6 alkoxy carbonyl group, hydroxyl group, hydroxy C.sub.1-6
alkyl group, substituted or non-substituted carbamoyl group,
alkyl-substituted or non-substituted amino group, acylamide group, and
alkoxylcarbonylamino group.
Alkyl group, alkenyl group, alkinyl group, and alkoxy groups may have
either straight chains or branches. Where an unsaturated bond exists, it
may be located in any arbitrary position. The halogen atom may be any one
of fluorine atom, chlorine atom, bromine atom and iodine atom. Specific
examples of the p-substituted phenol compounds include 4-methoxyphenol,
4-ethoxyphenol, 4-iodophenol, 3-(4- hydroxyphenyl) propionic acid (HPPA),
4-hydroxyphenylacetic acid, 4-hydroxyhippuric acid, p-cresol and tyramine.
Examples of p-substituted phenol compounds preferably used in the novel
method include the p-substituted phenol compounds given by the above
structural formula (2), where R.sup.1 is a hydrogen atom or c.sub.1-6
alkyl group, R.sup.2 is C.sub.1-6 alkyl group, carboxyl, or C.sub.1-6
alkoxy carbonyl group. Among them, especially preferable compounds include
the above-described compounds where R.sup.1 is a hydrogen atom and
compounds where R.sup.2 is CH.sub.3, C.sub.2 H.sub.5, C.sub.3 H.sub.7,
COOH, OCH.sub.3 or OC.sub.2 H.sub.5.
Examples of substances to be detected by the novel method include
peroxidase and equivalent catalytic substances such as hemoglobin.
Although the measuring method will be described in detail below, the
concentrations of reagents, the method of measuring ESR spectra and the
ESR spectroscopy are not limited to the illustrated examples. Obviously,
appropriate changes and modifications may be made to the illustrated
method and apparatus and those skilled in the art can make appropriate
choices.
EXAMPLE 1
Production of Reagent for Detection of Peroxidase or Hydrogen Peroxide
(1) After dissolving 1 g of carboxy-PTIO
›2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, 4.99
mmol, produced by Dhojin Chemical Laboratory Inc., Japan! in 5 ml of
methanol, ascorbic acid of twice molar quantity was added to the mixture
at room temperature. The liquid was stirred until white precipitation
appeared. The produced white precipitation was filtered out, cleaned with
a small amount of methanol and water and dried. As a result, a reagent of
carboxy-PTIOH
›2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-N-hydroxy-3-oxide!,
used for detection of peroxidase or hydrogen peroxide, was obtained.
(2) After dissolving 200 mg of carboxy-TEMPO
(4-carboxy-2,2,6,6-tetramethyl-1-piperidinyloxy, 7.21 mmol, prepared by
Aldrich Corporation) in 2 ml of methanol, ascorbic acid of twice molar
quantity was added to the mixture at room temperature. The liquid was
stirred until white precipitation appeared. The produced white
precipitation was filtered out, cleaned with a small amount of methanol
and water and dried. As a result, a reagent of
4-carboxy-2,2,6,6-tetramethyl-1-N-hydroxypiperidine, used for detection of
peroxidase or hydrogen peroxide, was obtained.
EXAMPLE 2
Detection of Peroxidase
As a buffer solution for detection, 0.1M MOPS buffer solution having a pH
of 6.5 and containing 0.015% H.sub.2 O.sub.2 was used. As a substrate
compound, p-acetamide phenol (25 mM, aqueous solution) was used. As a
hydroxy amine compound that is a reagent used for detection of peroxidase,
50 .mu.g/ml DMSO solution of
4-hydrazonomethyl-1-hydroxy-2,2,5,5-tetramethyl-3-imidazoline-3-oxide
(HHTIO) (Aldrich Corporation) or 50 .mu.g/ml DMSO solution of hydroxy
amine compound (carboxy-PTIOH) of Example 1 or 2 was used. As an enzyme
solution, 10.sup.-4 units/ml of peroxidase solution was prepared with
0.01M DIPSO buffer solution having a pH of 6.5.
100 .mu.l of the buffer solution for detection, 110 .mu.l of extra pure
water, 30 .mu.l of the substrate compound, 10 .mu.l of the hydroxy amine
compound, and 50 .mu.l of the enzyme solution are mixed. The mixture was
caused to react with each other at room temperature for 15 minutes. The
amounts of produced nitroxide radical species were measured by ESR
spectroscopy. The results are shown in FIGS. 1(a) and 1(b). FIG. 1(a)
shows the results of detection of carboxy-PTIOH. FIG. 1(b) shows the
results of detection of HHTIO. The top of signal lines indicates the
results of detection of a blank. As can be seen from these results, where
the p-substituted phenol was added, the ESR signal intensity was much
greater than the signal obtained from the blank to which no peroxidase was
added. The ESR measurements were made under the following conditions: the
center magnetic field strength was 3346.+-.50 Gauss; modulation was made
at 100 kHz and 1.0 Gauss; the power was 20 mW; the gains were 1.times.100
and 5.times.10, respectively; and the response was 0.1 sec.
EXAMPLE 3
Detection of Peroxidase, Using P-Hydroxyacetanilide Derivative
P-hydroxyacetanilide derivatives, p-methoxy phenol, p-cresol, thyroxine,
and 3-(4-hydroxyphenyl) propionic acid (HPPA) were used as p-substituted
phenol derivatives. HHTIO was used as a hydroxy amine compound, which is a
detection reagent. Using these compounds, peroxidase was quantitatively
detected. As a buffer solution for the detection, 0.1M MOPS buffer
solution having a pH of 6.5 and containing 0.015% H.sub.2 O.sub.2 was
used. As a substrate solution, an aqueous solution (25 mM) was used. As an
enzyme solution, 10.sup.-4 units/ml of POD solution was adjusted with
0.01M MOPS buffer solution having a pH of 7.0. As an HHTIO solution, 50
.mu.g/ml solution was prepared with DMSO.
50 .mu.l of the POD solution to 100 .mu.l of the buffer solution, 30 .mu.l
of the substrate solution, 120 .mu.l of H.sub.2 O.sub.2, and 10 .mu.l of
the HHTIO solution are mixed. The mixture was caused to react with each
other at room temperature for 30 minutes. The amounts of the produced
radicals were measured with an ESR spectrometer JES-FR80 manufactured by
JEOL Ltd., Japan, under the following conditions: the center magnetic
field strength was 3346.+-.50 Gauss; modulation was made at 100 kHz and
1.0 Gauss; the power was 20 mW; the gains were 1.times.100 and 5.times.10,
respectively; and the response was 0.3 sec. The results are listed in
Table 1 below. In Table 1, the efficiency of enhancement of the
sensitivity is the peak height/blank. It can be seen that the
p-hydroxyanilide derivatives showed higher efficiencies of enhancement of
the sensitivity than other phenolic compounds.
TABLE 1
______________________________________
p-substituted
peak height
efficiency of enhancement
phenol (mm) of sensitivity
______________________________________
blank 3.0 --
R.sup.1 = H, R.sup.2 = CH.sub.3
610 203.3
R.sup.1 = H, R.sup.2 = C.sub.2 H.sub.5
625 208.3
R.sup.1 = H, R.sup.2 = C.sub.3 H.sub.7
580 193.3
R.sup.1 = H, R.sup.2 = COOH
60 20.0
R.sup.1 = H, R.sup.2 = OCH.sub.3
710 236.8
R.sup.1 = H, R.sup.2 = OC.sub.2 H.sub.5
592 197.3
p-methoxyphenol
405 135.0
p-cresol 125 41.7
thyroxine 10.0 3.3
HPPA 18.0 6.0
______________________________________
EXAMPLE 4
Comparison of the Novel Method with Prior Art Method, Using Color-Producing
Reagents ABTS and OPD
The sensitivity of the novel method was compared with the sensitivity of
the prior art method, using ABTS ›2,2-azino-bis
(3-ethyl-benzothiazoline-6-sulfonic) acid! and OPD (o-phenylene diamine)
which are typical color-producing reagents used for detection of
peroxidase.
For the measurements with ABTS, a substrate buffer solution and a
color-producing reagent attached to Lanaenzyme 439 kit manufactured by
Nippon Kayaku Co., Ltd., Japan, where used, and 300 .mu.l of the substrate
solution whose ABTS concentration was adjusted to 2.5 mg/ml was employed.
For the measurements with OPD, an OPD tablet-dissolving solution attached
to HBs antigen-measuring reagent AUSZYME II prepared by Abbot Inc. were
used. 5 ml of the OPD tablet-dissolving solution was added per tablet. 300
.mu.l of the substrate solution whose OPD concentration was adjusted to 3
mg/ml was used. 50 .mu.l of peroxidase solution (10-5 u/ml) was added to
300 .mu.l of each substrate solution. Both mixtures were caused to react
at room temperature for 30 minutes. For the measurements with ABTS, the
reaction was quenched or brought to a stop by adding 1000 .mu.l of H.sub.2
O.
For the measurements with OPD, 1000 .mu.l of 1N H.sub.2 SO.sub.4 was added
for the same purpose. The final volume of the final product was controlled
to 1000 .mu.l. The OD values were measured, using a spectrophotometer
U-2000 manufactured by Hitachi Ltd., Japan. Wavelengths of 414 nm and 490
nm, respectively, were used for the measurements, using ABTS and OPD,
respectively.
Measurements by the novel method using hydroxy amine compounds were carried
out similarly to the measurements of Example 3 except that the amount of
ultra pure water was 160 .mu.l and that the gain was .times.200. The
results are listed in Table 2. The results of measurements by the novel
method are shown in FIGS. 2(a) and 2(b). FIG. 2(a) shows the results of
the measurement using carboxy-PTIOH. FIG. 2(b) shows the results of the
measurement using HHTIO. The top lines indicate the results of
measurements using a blank. The signal-to-noise ratios (S/N) where ABTS
and OPD were used were 3.0 and 3.3, respectively. In the novel method,
where HHTIO was used, the S/N of the obtained ESR signal was 47.6. Where
PTIOH was used, the S/N was 15.3. In this way, the measuring method
according to the invention provides much higher sensitivity than the prior
art method.
TABLE 2
______________________________________
specimen ABTS (414 nm)
OPD (490 nm)
inventive (mm)
______________________________________
blank 0.004 0.003 2.5
POD 10.sup.-5 u/ml
0.012 0.010 11
______________________________________
EXAMPLE 5
Detection of Hydrogen Peroxide H.sub.2 O.sub.2
100 .mu.l of DIPSO buffer solution (0.1M, pH: 7.0), 30 .mu.l of p-acetamide
phenol (20 mmol/L), 10 .mu.l of HHTIO (1 mmol/L), and 50 .mu.l of
peroxidase solution (10.sup.-1 units/ml) was added to 100 .mu.l of H.sub.2
O.sub.2 diluted solution (concentration %). They were made to react with
each other at room temperature for 10 minutes. Then, the amounts of
produced radicals were measured by an ESR spectrometer. The amount of the
peroxidase was excessive for H.sub.2 O.sub.2. Measurements were made with
five concentrations of the H.sub.2 O.sub.2 diluted solution (i.e.,
0.625.times.10.sup.-6 %, 1.25.times.10.sup.-6 %, 2.5.times.10.sup.-6 %,
5.0.times.10.sup.-6 %, and 10.times.10.sup.-6 %). The results of the
measurements are shown in FIGS. 3 and 4. FIG. 3 shows ESR signal waveforms
at these various concentrations. FIG. 4 is a graph in which the relation
of the concentration to the ESR signal intensity is plotted. As can be
seen from FIG. 4, a proportional relation exists between the ESR signal
intensity and the concentration of hydrogen peroxide. Detection of
hydrogen peroxide at the concentration of 1.25.times.10.sup.-6 % was
possible. The ESR measurements were made under the following conditions:
the center magnetic field strength was 337.500 mT; modulation was made at
100 kHz and 0.1 mT; the power was 10 mW; the gain was .times.200; and the
response was 0.1 sec.
EXAMPLE 6
Application to Biochemical Tests
Example 5 has demonstrated that quantitative measurement of H.sub.2 O.sub.2
is possible under a condition that peroxidase is overabundant to H.sub.2
O.sub.2. There are many biochemical tests based on quantitative
measurements of H.sub.2 O.sub.2 produced by an enzyme reaction. Such tests
are directed to measurements of glucose, lactic acid, pyruvic acid, sialic
acids, uric acid, creatinine, polyamine, total cholesterol, free
cholesterol, neutral fat, phospholipid, free aliphatic acid, inorganic
phosphorus, etc. Accordingly, it is possible to adopt the novel method in
such tests by quantitatively measuring H.sub.2 O.sub.2 produced by an
enzyme reaction.
As one example of application of the present invention to biochemical
tests, a quantitative measurement of glucose is described below.
100 be of MOPS buffer solution (0.1M, pH: 6.5), 50 .mu.l of p-acetamide
phenol (20 mmol/L), 1 .mu.l of a standard substance (100, 200 or 400
mg/dL) or monitored sera (Moni-Trol I & II, Baxter Diagnostic Inc.), 10
.mu.l of HHTIO (3 mmol/L), 50 .mu.l of peroxidase solution (10.sup.-1
units/ml) and 50 .mu.l of glucose oxidase (5/units/ml) were mixed. The
mixture was stirred and then caused to react at 37.degree. C. for 10
minutes. A reaction-stopping agent, or 50 .mu.l of NaN.sub.3 (500 mmol/L),
was added. Thereafter, measurements were made by ESR spectroscopy.
FIG. 5(a) shows ESR signal waveforms derived from the standard substance.
FIG. 5(b) shows ESR signal waveforms obtained from the monitored sera
Moni-Trol I & II. The values of the sera Moni-Trol I & II found, based on
the standard substance, are 80 mg/dL and 235 mg/dL, respectively, which
well agree with the values denoted on the containers of the sera.
According to the present invention, peroxidase or hydrogen peroxide can be
detected with high sensitivity. Also, analysis of peroxidase which is so
dilute that it cannot be detected by the prior art method is enabled.
Having thus described our invention with the detail and particularity
required by the Patent Laws, what is desired protected by Letters Patent
is set forth in the following claims.
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